The proto-oncogene encoding the Tpl-2 kinase, is activated by provirus integration in retrovirus-induced rodent lymphomas and mammary adenocarcinomas. Overexpression of Tpl2 in a variety of cell types activates ERK, JNK, p38 MAPK, and the transcription factors NF-AT and NF-KB. Moreover, transgenic mice expressing a constitutively-active form of Tpl-2 under the control of a T cell specific promoter develop thymic lymphomas. Finally, Tpl2 contributes to the transduction of oncogenic signals induced by LMP1 and MyrAkt1. Our studies using Tpl2 knockout mice revealed that Tpl2 plays an obligatory role in the transduction of Toll-like receptor and death receptor signals. Ablation of Tpl2 in macrophages interferes only with signals that activate ERK, while ablation of Tpl2 in mouse embryo fibroblasts interferes with signals that activate ERK, JNK and NF-KB. Because of these signaling defects, Tpl-2 knockout mice are resistant to LPS/D-galactosamine and high dose LPS-induced shock as well as to TNF-alpha induced arthritis and inflammatory bowel disease. In unstimulated macrophages, Tpl2 is stoichiometrically bound to NF-KB 1. LPS stimulation promotes the dissociation of the two proteins. Unbound Tpl2 is active but unstable, undergoing rapid degradation via the proteasome. The proposed experiments will address the molecular mechanisms that regulate Tpl2 in response to LPS or TNF-alpha signals. These experiments will be based on several observations we have recently made: a) Tpl2 is phosphorylated at Thr290 in response to LPS. Phosphorylation promotes the LPS-induced dissociation of Tpl2 from NF-KB 1 and the activation of the Tpl2 kinase; b) Whereas wild type Tpl2 undergoes ubiquitination, kinase-inactive Tpl2 does not. Proteasome inhibitors increase the stoichiometry of Tpl2 ubiquitination and enhance signaling pathways activated by Tpl2. Finally, Tpl2 binds the E3 ubiquitin ligases TRAF6 and TRAF2 and is required for the transduction of TRAF6, and perhaps TRAF2-induced signals. These data combined, indicate that ubiquitination may contribute to Tpl2 activation by external signals. Based on this information, we designed experiments that utilize molecular genetics, tissue culture technologies to address the mechanisms of Tpl2 activation by LPS and TNF-alpha.